Collagen breakdown in periodontal disease presumably is initiated by secretion of collagenase by the transient and/or permanent gingival cell populations. However, the lack of sensitive analytical methods has precluded a definition of the precise role of collagenase in the disease. This proposal has as its major objective the development and analytical use of methods to quantitate and localize collagenase. The enzyme will be partially purified and used to produce mouse monoclonal antibodies. Collagenase will then be completely purified by immuno-affinity chromatography and preparative acrylamide electrophoresis and employed to raise monospecific, precipitating anitbodies in rabbits. Single- and double-labeling immunofluorescent staining procedures will be establshed and electron microscopic procedures to localize collagenase with ferritin-conjugated Ig will be developed. Soluble- and solid-phase radioimmunodisplacement assays (RIDA) using 125I-collagenase will be developed and procedures will be established to combine quantitative rock immuno-electrophoresis with autoradiography. These methods will be employed (1) to identify collagenase in transient and permanent periodontal cells; (2) to study the subcellular and extracellular localization of collagenase; and (3) to quantitate collagenase in gingival cell and tissue homogenates, and in crevicular fluids.